PROCESS FOR THE PREPARATION OF HYPOALLERGENIC MAJOR BIRCH POLLEN ALLERGEN rBet v 1

ABSTRACT

The invention relates to a process for the preparation of hypoallergenic major birch pollen allergens by means of one or more chromatographic purification steps using essentially unbuffered aqueous bases as eluent and subsequent neutralisation. The hypoallergenic major birch pollen allergens are distinguished by a lack of, but at least by reduced immunoglobulin E binding with simultaneous retention of therapeutically relevant T-cell stimulation. They can therefore be employed as low-side-effect therapeutic agents for specific immunotherapy.

TECHNICAL AREA OF THE INVENTION

The invention relates to a process for the preparation of birch pollenallergens which are distinguished by a lack of, but at least by reducedimmunoglobulin E binding, i.e. by hypoallergeneity. These allergenscompletely retain therapeutically relevant T-cell stimulation. They cantherefore be employed as low-side-effect therapeutic agents for specificimmunotherapy.

BACKGROUND OF THE INVENTION

Type 1 allergies have dramatically increased worldwide in recentdecades. Up to 20% of the population in industrialised countries sufferfrom complaints, such as allergic rhinitis, conjunctivitis or bronchialasthma, which are caused by allergens present in the air(aeroallergens), which are released by various sources, such as plantpollen, mites, mammals (cats, dogs, horses) and mould fungi. Severeallergies can also be initiated by insect stings or bites, such as, forexample, of bees and wasps.

The type 1 allergy-initiating substances are proteins, glycoproteins orpolypeptides. These allergens react via the mucous membranes afteringestion or with the IgE antibodies bound to the surface of mast cellsin sensitised people after stings or bites. If two or more IgEantibodies are crosslinked to one another by an allergen, this resultsin the release of mediators (for example histamine, prostaglandins) andcytokines by the effector cell and thus in initiation of the allergicsymptoms.

Birch pollen are the most frequent initiators of allergic reactionsamongst tree pollen (Jarolim E. et al., 19891 Allergy 44:385-95). Morethan 90% of sufferers from birch pollen allergies have IgE antibodiesagainst the major allergen Bet v 1 (Elfman, L. et al., 1997, Int, Arch.Allergy Immunol., 113: 249-51).

With the aid of cDNA sequences, it is possible to prepare recombinantallergens which can be used in the diagnostics and therapy of allergies.(Scheiner and Kraft, 1995, Allergy 50, 384-391). The preparation ofrecombinant Bet v 1 allergens (rBet v 1) and their purification forpharmaceutical purposes has been described, for example, byHoffmann-Sommergruber et al. (Protein Exp. Purint 9 (1), 1997: 33-39).

In addition, specific genetic modification of recombinant allergens ispossible, enabling a reduced allergenic potential to be achieved(Schramm et al. 1999, J. Immunol. 162 (4); 2406-2414; Valenta et al.,1999, Biol. Chem. 380: 815-24; Singh et al., 1999, Int. Arch. AllergyImmunol. 119: 75-85). Allergen variants of this type are promisingfuture candidates for specific immunotherapy of type 1 allergy.

However, a potential disadvantage in recombinant allergen variants isthat the modification of the primary structure causes loss or areduction in the reactivity of the T-cell epitopes which are necessaryfor therapeutic success. This possibility can only be excluded if theprimary structure corresponding to the natural allergen serves as thebasis for the preparation of the recombinant protein.

In the case of major birch pollen allergen Bet v 1, the preparation wascarried out in two halves (Vrtala, S., et al., 1997, J, Clin. Invest.99: 1673-81) or as a trimer (Vrtala, S., et al., 1999, Int. Arch.Allergy Immunol. 118:218-9) in order to optimise it for therapeuticpurposes, i.e. to reduce the IgE binding capacity, by recombinantmethods. The potential loss of T-cell epitopes and the insolubility ofthe proteins also has a disadvantageous effect in these approaches. Afurther disadvantage here can be seen in the complex manner ofpreparation of these rBet v 1 variants.

A suitable starting point for the preparation of a recombinant majorallergen rBet v 1 which can be utilised for therapeutic purposes wouldaccordingly be a molecule which corresponds to the wild type in theprimary structure and is unrestricted in its T-cell stimulation, but hasreduced IgE activity, i.e. is hypoallergenic.

This object has been achieved in accordance with the present inventionby the performance of a series of biochemical purification steps knownper se using soluble recombinant major allergen rbet v 1 as startingmaterial, Surprisingly, a reduced IgE activity and at the same timemaintained T-cell stimulation has been observed in the proteins purifiedin this way. Accordingly, the process according to the inventionprovides improved therapeutic efficacy at the same time as a significantreduction in or absence of side effects.

The form of the preparation process of the recombinant allergens is ofparticular importance here inasmuch as the proteins are converted in thecourse of this process into a conformation which has no or greatlyreduced affinity to IgE with constant T-cell stimulation.

FIGURES

FIG. 1A: SDS-PAGE for characterisation of hypoallergenic rBet v 1

Track 1: Protein standard for estimation of the molecular weight

Track 2: Natural rBet v 1

Track 3: rBet v 1 purified in accordance with the invention

Track 4, Conventionally purified rBet v 1

FIG. 1B: Nitrocellulose blot of the SDS-PAGE from FIG. 1A

FIG. 2A: Nitrocellulose blot for determination of the IgE activity with20 individual patient sera

Position 3: Natural rBet v 1

Position 4: rBet v 1 purified in accordance with the invention

Position 5: Conventionally purified rBet v 1

FIG. 28: Nitrocellulose blot for determination of the identity of Bet v1 samples

Blots 21-26: Various polyclonal rabbit anti-Bet v 1 antibodies

Blot 27: Monoclonal mouse anti-Bet v 1 antibody 6B6

FIG. 3: Enzyme allergo sorbent test (EAST) for quantification of IgEbinding

The concentration of an inhibitor of IgE-Bet v 1 binding in μg/ml isplotted on the vertical axis, and the degree of inhibition in [%] isshown on the horizontal axis.

FIG. 4: Determination of T-cell stimulation by Bet v 1 variants

The concentrations of natural rBet v 1, of conventionally purifiedrecombinant rBet v 1 and of recombinant rBet v 1 purified in accordancewith the invention and the respective stimulation indices (St) obtainedwith various T-cell lines (TCLs) and T-cell clones (TCCs) are compared.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a biochemical purification processwhich results in the preparation of proteins having the propertiesmodified in accordance with the invention via efficient purification,using specific eluents, of, for example, allergens prepared byrecombinant methods. These properties consist in a lack of, but at leasta greatly reduced IgE activity, with simultaneous maintenance of T-cellstimulation.

The invention thus relates to a process for reducing the IgE activity ofthe major birch pollen allergen rbet v 1 which consists in the use ofsoluble recombinant major birch pollen allergen rbet v 1 and in carryingout the chromatography steps described below for the purificationthereof and the subsequent neutralisation step.

The invention furthermore relates to a process for the preparation ofhypoallergenic major birch pollen allergen rBet v 1 by means of aplurality of chromatographic purification steps using essentiallyunbuffered aqueous bases as eluent and subsequent neutratisation, wherethe starting material employed is a soluble rBet v 1 crude proteinprepared by recombinant methods.

The chromatographic purification steps preferably include anion exchangechromatography, hydrophobic interaction chromatography and gelfiltration and can be carried out once, a number of times one after theother or a number of times alternately. However, the chromatographicpurification steps are preferably carried out in the following sequence:first gel filtration, anion exchange chromatography, hydrophobicinteraction chromatography, second get filtration.

The chromatographic purification is usually carried out with a baseconcentration of from 5 to 100 mM, but preferably with from 5 to 40 mMand particularly preferably with from 10 to 30 mM, where the basicsubstance employed is preferably NaOH. Instead of a purely aqueoussystem, it is also possible to use a mixed system comprising, forexample, water and methanol. A non-aqueous, for example methanol systemis also conceivable. However, preference is given to the use of anaqueous system

Depending on the respective chromatography step, differentconcentrations of a neutral salt—preferably NaCl—up to about 5 M can beadded to the eluent.

Any reduction in the pH that may be necessary to values which aretolerated by relatively sensitive proteins can be achieved by theaddition of sodium hydrogencarbonate.

The intention of establishing physiological conditions at the end of thepurification can also be a reason for the presence of sodiumhydrogencarbonate during the chromatography steps. Concentrations of upto 100 mM are basically possible here. However, work is preferablycarried out in the physiological range below 20 mM, particularlypreferably at 11 mM.

In the case of rbet v 1, a reduction in the pH is not necessary.Nevertheless, sodium hydrogencarbonate is generally added on applicationof the process according to the invention to rbet v 1 in order to beable to establish physiological concentrations in a simple manner at theend of the purification.

The invention thus relates to a process for the preparation ofhypoallergenic major birch pollen allergen rBet v 1 in which essentiallyunbuffered basic eluents maintain the proteins to be purified insolution under mild conditions and thus in a chromatographable state.

In a preferred embodiment of the process, the rBet v 1 crude protein ispre-purified before the actual purification by chromatography, forexample hydrophobic interaction chromatography or ion exchangechromatography, and/or salt precipitation, where, in contrast to thesubsequent principal purification, a buffered eluent or a bufferedsolution is used.

The starting materials for the process are soluble, recombinantallergens expressed in bacteria or other suitable host cells (such as,for example, yeasts). Since the process represents a general applicationfor these expression products, soluble allergens of different origin canalso, in accordance with the invention, be purified by the process,renatured and formulated. In particular in the case of correspondingsimilarity of these allergens of different origin to the major birchpollen allergen Bet v 1, it can be expected that the propertiesaccording to the invention will be achieved. However, the process isparticularly suitable for obtaining major birch pollen allergen rbet v 1prepared by recombinant methods. However, natural major birch pollenallergen rBet v 1 is also basically suitable as starting material.

Final neutralisation of the recombinant active ingredients by modifyingthe pH enables—given a corresponding choice of concentration of theeluent ingredients—a ready-to-use physiological solution to be obtaineddirectly.

The pharmaceutical active ingredients can be used directly afterneutralisation as parenteral products. In addition, the reproducible andstandardisable process can be carried out under the conditions of GoodManufacturing Practice (GMP), which is necessary for pharmaceuticals.

The invention thus serves for the preparation of improved preparationsfor the specific immunotherapy of allergies, which is achieved by theprocess according to the invention. The greatly reduced or lack of IgEactivity provides advantageous properties for specific immunotherapy.Recombinant hypoallergenic allergens prepared in this way can thuscontribute to improved therapy of allergic diseases.

The invention therefore relates to a hypoallergenic major birch pollenallergen rBet v 1 obtainable by the process according to the invention,in particular in its use as medicament.

It is of course possible, for additional influencing of the propertiesof major birch pollen allergen rBet v 1—for example in order to achievea further reduction in the IgE activity or a further increase in T-cellstimulation—to make pharmaceutically acceptable modifications to theprotein and thus to produce derivatives of the protein. Thesemodifications can on the one hand be genetic modifications at the DNAlevel, where, for example, amino acid insertions, deletions andreplacements, cleavage of the protein into fragments and fusion of theprotein or fragments thereof with other proteins or peptides aresuitable. However, the modifications can also be of a chemical natureand take place at the protein level.

The invention thus relates to the use of hypoallergenic major birchpollen allergen rBet v 1 according to the invention and/orpharmaceutically usable derivatives thereof, including mixtures thereofin all ratios, for the preparation of a medicament for the specificimmunotherapy of allergies in the initiation of which major birch pollenallergen Bet v 1 is involved. Finally, the invention relates to apharmaceutical composition comprising hypoallergenic major birch pollenallergen rBet v 1 according to the invention and/or pharmaceuticallyusable derivatives thereof, including mixtures thereof in all ratios.The active ingredients according to the invention can be converted hereinto a suitable dosage form together with at least one solid, liquidand/or semi-liquid excipient or adjuvant and optionally in combinationwith one or more further active ingredients.

These compositions can be used as therapeutic agents in human orveterinary medicine. Suitable excipients are organic or inorganicsubstances which are suitable for parenteral administration and do notreact with hypoallergenic major birch pollen allergen rBet v 1. Suitablefor parenteral administration are, in particular, solutions, preferablyoil-based or aqueous solutions, furthermore suspensions, emulsions orimplants. Hypoallergenic major birch pollen allergen rBet v 1 accordingto the invention can also be lyophilised and the resultant lyophilisatesused, for example, for the preparation of injection preparations. Thecompositions indicated can be sterilised and/or comprise adjuvants, suchas lubricants, preservatives, stabilisers and/or wetting agents,emulsifiers, salts for modifying the osmotic pressure, buffer substancesand/or a plurality of further active ingredients.

Furthermore, corresponding formulation of hypoallergenic major birchpollen allergen rBet v 1 according to the invention enables depotpreparations to be obtained, for example through adsorption ontoaluminium hydroxide.

The preparation process according to the invention is described ingeneral form below. In this description, all chromatography materialsmentioned by way of example come from Amersham Biosciences (Freiburg,Germany). A first pre-purification step for removal of nucleic acids canconsist of hydrophobic interaction chromatography carried out underphysiological conditions (at a pH of 6-8, non-denaturing), where thetarget protein is simultaneously focused. Alternatively, saltprecipitation or ion exchange chromatography can also be carried out.However, it is not absolutely necessary to carry out this firstpre-purification step for achieving the effect according to theinvention.

The next purification step serves to convert the proteins into weaklysaline eluent in the concentration range 10-100 mM, for example 20 mMNaCl, for example by means of gel-filtration on a Sephadex G-25 column.Conditions are thus created which facilitate the performance of ionexchange chromatography using alkaline eluents. The protein solutionprepared in this way is subsequently employed for anion exchangechromatography, for example using a Source Q column. Most allergens arebound to the support here. The alkaline eluent causes even previouslysparingly soluble or insoluble proteins to remain in solution. NaClgradient elution results in partial removal of bacterial impurities andactive-ingredient fragments.

In two further purification steps, hydrophobic interactionchromatography and gel filtration, the pre-purified and equilibratedallergens are essentially separated from bacterial impurities stillremaining. To this end, use is basically made of the same eluentsubstances consisting of low-molar base and a varying proportion of aninorganic neutral salt. Thus, the allergens can be bound to the columnin hydrophobic interaction chromatography using, for example, up to 5 MNaCl, 20 mM NaOH and 11 mM NaHCO₃ and subsequently eluted using low-saltor salt-free alkaline solution, for example 20 mM NaOH.

In the final chromatography step, an eluent change is carried out insuch a way that the purified recombinant proteins are obtained insoluble, ready-to-use form by simple neutralisation of the base presentin the eluent using a corresponding acid. Given a suitable choice of theconcentrations of the eluent additives, a physiological solution whichis suitable for parenteral products is formed.

The purified allergens are identified via their known physical, chemicalor biological properties, in particular by means of SDS-PAGE andspecific monoclonal antibodies. For further characterisation, an EASTinhibition assay (EAST denotes enzyme allergo sorbent test), with whichthe specific IgE binding of a protein compared to a reference cane bedetermined, and/or a T-cell proliferation assay, for example, can becarried out.

The solvent is tested by pH measurement and quantification of the Na⁺and Cl⁻ and, if desired, CO₃ ²⁻ concentration. These methods aregenerally known and described.

The yield of the allergens prepared in accordance with the invention isgenerally 75-95%, based on the starting protein.

The process thus involves minimal sample treatments, short samplestanding times, preferably the use of exclusively pharmacologicallycompatible substances, compatibility of a single eluent with diverseseparation principles, and the avoidance of lengthy and under certaincircumstances non-validatable methods, such as dialysis, in addition,the sodium hydroxide solution preferably employed as base, which isknown as an effective bacteriostatic, prevents the proteins presenttherein from being degraded or contaminated by microorganisms.Endotoxins, which can cause problems in bacterial expressions, otherforeign proteins and DNA are likewise effectively removed or degraded.

The sequence and number of chromatography steps described above can bechanged. Thus, inter alia, the specific physicochemical properties ofthe target proteins can be taken into account.

Even without further comments, it is assumed that a person skilled inthe art will be able to utilise the above description in its broadestscope. The preferred embodiments described below should therefore merelybe regarded as descriptive disclosure which is absolutely not limitingin any way.

A particularly preferred embodiment of the process is shown in thefollowing scheme (Tab. 1):

TABLE 1 Overview of the preparation process according to theinvention 1. Pre-purification (optional) hydrophobic interactionchromatography (phenyl-Sepharose) Eluent 1: 20 mM Tris/HCl, 1 M ammoniumsulfate, pH 8.0 Eluent 2: dist. water 2. Eluent change for the mainpurification gel filtration (Sephadex 25) Eluent 20 mM NaCl 3. Anionexchange chromatography (Source 15Q) Eluent 1: 20 mM NaOH, 11 mM NaHCO₃and 20 mM NaCl Eluent 2: NaCl gradient (from 20 mM NaOH; 11 mM NaHCO₃;      20 mM NaCl to 20 mM NaOH; 11 mM NaHCO₃;       0.5 M NaCl) 4.Hydrophobic interaction chromatography (Source PHE) Eluent 1: 3 M NaCl,20 mM NaOH, 11 mM NaHCO₃ Eluent 2: 20 mM NaOH 5. Gel filtration(Superdex 75) Eluent: 10 mM NaOH, 11 mM NaHCO₃ and 148.4 mM NaCl 6.Neutralisation using 1/10 (v/v) 100 mM HCl

The invention is described below through the example of the purificationof therapeutically effective recombinant Bet v 1 (rBet v 1). Allchromatography materials are commercially available from AmershamBiosciences (Freiburg, Germany).

EXAMPLE 1 Preparation of Hypoallergenic rBet v 1

Firstly, an E. coli lysate containing soluble rBet v 1 allergen isprepared by standard methods (Breiteneder H., et al., EMBO J. 1989, 8:1935-8; Hoffmann-Sommergruber et al., Protein Exp. Purif. 9 (1), 1997:33-39).

In order to remove nucleic acids, hydrophobic interaction chromatographyis then carried out using phenyl-Sepharose in a Tris/ammonium sulfatebuffer (20 mM Tris/HCl, 1 M ammonium sulfate, pH 8.0). The elution iscarried out with distilled water. In preparation for the furtherpurification steps to be carried out with weakly alkaline eluents, theresidual ammonium sulfate is replaced by 20 mM NaCl by means of gelfiltration through Sephadex G-25.

The protein solution pre-purified in this way is employed for anionexchange chromatography using Source 15Q, where the support material isequilibrated with an alkaline solution (20 mM NaOH, 11 mM NaHCO₃ and 20mM NaCl). The relatively high pH of the starting solution causesvirtually all target proteins to bind to the anion exchanger. Thesubsequent elution is carried out with increasing NaCl gradient (from 20mM NaOH; 11 mM NaHCO₃; 20 mM NaCl to 20 mM NaOH; 11 mM NaHCO₃; 0.5 MNaCl) and causes removal of impurities (host-cell proteins) andactive-ingredient fragments.

The next chromatography step is hydrophobic interaction chromatographyby means of Source PHE. To this end, the eluate from the ion exchangechromatography is adjusted to 3 M NaCl, 20 mM NaOH, 11 mM NaHCO₃ byaddition of corresponding amounts of a 5 M NaCl stock solution, a 2 MNaOH stock solution and sodium hydrogencarbonate. Under theseconditions, rBet v 1 binds to the column material. The elution of thebound target protein is carried out using 20 mM NaOH.

As the final step, gel filtration is carried out through Superdex 75under alkaline conditions. The chromatography solution is selected insuch a way that neutralisation of the base added to the eluent resultsin the desired final formulation: 10 mM NaOH, 11 mM NaHCO₃ and 148.4 mMNaCl, which corresponds to the concentrations of physiological salinesolution. The eluate from the gel filtration is finally neutralisedusing the acid HCl corresponding to the base NaOH used, resulting in aneutral pH and at the same time achieving the desired salt content ofphysiological saline solution. This is achieved through addition of 1/10(v/v) 100 mM HCl.

EXAMPLE 2 Characterisation by SDS-PAGE

For characterisation of hypoallergenic rBet v 1 from Example 1, anSDS-PAGE (15%) is carried out. As can be seen from FIG. 1A, the naturalnBet v 1 (track 2), recombinant rBet v 1 purified in a conventionalmanner by the method of Hoffmann-Sommergruber et al. (Protein Exp.Purif. 9 (1), 1997: 33-39) (track 4) and rBet v 1 purified in accordancewith the invention (track 3) have the same molecular weight in theSDS-PAGE.

EXAMPLE 3 Determination of the IgE Activity Using a Serum Pool

In order to determine the IgE activity, the SDS-PAGE from Example 2 isblotted on nitrocellulose. After a blood serum pool from birch pollenallergy sufferers has been added to the blot, the blot is incubated witha conjugate consisting of an anti-IgE antibody and alkaline phosphatase.The colour reaction promoted by the alkaline phosphatase (FIG. 1B) showsan IgE activity of natural nbet v 1 and of recombinant, conventionallypurified rBet v 1, but not of rBet v 1-MF purified in accordance withthe invention.

EXAMPLE 4 Determination of the IgE Activity Using Individual Sera

In order to determine the IgE activity using blood sera from individualbirch pollen allergy sufferers, nBet v 1 (position 3), recombinant,conventionally purified rBet v 1 (position 5) and rBet v 1 purified inaccordance with the invention (position 4) are applied, as shown in FIG.2A, to a nitrocellulose membrane and analysed analogously to Example 3,FIG. 2A shows that, with the exception of serum 5, where rbet v 1purified in accordance with the invention has weak IgE activity, onlynatural nBet v 1 and recombinant, conventionally purified rBet v 1, butnot rBet v 1 purified in accordance with the invention have IgEactivity.

In order to determine the identity of the allergens investigated, thenitrocellulose membrane was incubated with various polygonal rabbitanti-Bet v 1 antibodies (samples 21 to 26) and with monoclonal mouseanti-Bet v 1 antibody 6B6 (sample 27) and subsequently treatedanalogously to Example 3 (FIG. 2B).

EXAMPLE 5 Quantification of IgE Binding

In an EAST inhibition assay carried out by the method of Suck et al.(Int. Arch. Allergy Immunol. 2000; 121: 284-291) using anallergy-sufferer serum pool, natural nBet v 1, recombinant,conventionally purified rBet v 1 and rBet v 1 purified in accordancewith the invention are compared with one another with respect to thestrength of their IgE binding (FIG. 3). It is found that rBet v 1purified in accordance with the invention is reduced in IgE activity bymore than 100 times compared with the other Bet v 1 proteins.

EXAMPLE 6 Determination of T-Cell Stimulation

In order to determine the influence of the rBet v 1 allergen accordingto the invention on the growth of T-cells, a proliferation assay withT-cell lines (TCLs) and T-cell clones (TCCs) is carried out by themethod of Schramm et al. (1999, J. Immunol. 162 (4): 2406-2414) (FIG.4). It can be seen from a comparison of the stimulation indices (SI)that the T-cells of the investigated donors react at least as stronglywith rBet v 1 as with natural nBet v 1 or conventional purifiedrecombinant rBet v 1. Depending on the conditions selected, the reactionwith rBet v 1 even exceeds the reaction with nBet v 1 or rBet v 1 by upto one third.

1. Process for the preparation of hypoallergenic major birch pollenallergen rBet v 1 by means of one or more chromatographic purificationsteps using essentially unbuffered aqueous bases as eluent andsubsequent neutralisation, characterised in that the starting materialemployed is a soluble rBet v 1 crude protein prepared by recombinantmethods.
 2. Process according to claim 1, characterised in that thesoluble rBet v 1 crude protein prepared by recombinant methods wasexpressed in E. coli.
 3. Process according to claim 1 or, characterisedin that the chromatographic purification steps include anion exchangechromatography, hydrophobic interaction chromatography and gelfiltration.
 4. Process according to one or more of claim 1,characterised in that the chromatographic purification steps are carriedout in the following sequence: first gel filtration, anion exchangechromatography, hydrophobic interaction chromatography, second gelfiltration.
 5. Process according to claim 1, characterised in that thebase used as eluent is employed in a concentration range from 5 mM to100 mM, preferably from 5 mM to 40 mM.
 6. Process according to claim 1,characterised in that the base used as eluent is NaOH.
 7. Processaccording to claim 1, characterised in that an inorganic neutral saltand optionally NaHCO₃ are added to the eluent.
 8. Process according toclaim 1, characterised in that the inorganic neutral salt added to theeluent is NaCl.
 9. Process according to claim 6, characterised in thatthe concentrations of the substances NaOH, NaCl and NaHCO₃ present inthe eluent are selected in such a way that the typical concentrationsfor physiological saline solution can be set in the neutralisation stepfollowing the purification.
 10. Process according to claim 1,characterised in that the rBet v 1 crude protein is pre-purified in abuffered solution by chromatography and/or by salt precipitation.11.-13. (canceled)
 14. A method for the specific immunotherapy ofallergies involving major birch pollen allergen Bet v 1 comprisingadministering, to a subject in need thereof, a hypoallergenic majorbirch allergen bet v 1 or a pharmaceutical preparation thereof. 15.(canceled)